Kit for detecting drug concentration of usterina monoclonal antibody and preparation method thereof

Abstract

The invention discloses a kit for detecting the drug concentration of an usterinum monoclonal antibody and a preparation method of the kit. The kit comprises a microwell plate, a biotin-labeled anti-usterinum monoclonal antibody, a calibration product and a quality control product. The microwell plate is coated with a mouse anti-ulinobacteria monoclonal antibody, and the biotin-labeled anti-ulinobacteria monoclonal antibody is obtained by modifying biotin on an antibody, so that the amplification of a detection signal is further improved, and the detection sensitivity is improved. And the calibrator comprises seven types of usterinum monoclonal antibody solutions with different concentrations. The quality control products are two types of ulinus monoclonal antibody solutions with calibrated concentrations. According to the kit for detecting the drug concentration of the usterinum monoclonal antibody, a double-antibody sandwich enzyme-linked immunosorbent assay is adopted, and the kit for detecting the drug concentration of the usterinum monoclonal antibody prepared by the preparation method disclosed by the invention has the advantages of small sample dosage, wide linear range and high sensitivity when being used.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibody detection, and particularly relates to a ustekinumab drug concentration detection kit and a preparation method. BACKGROUND

[0002] Ustekinumab is a monoclonal therapeutic antibody targeting p40, the common subunit of interleukin 12 and 23 (IL-12 / 23). These two cytokines play a fundamental role in immune-mediated inflammatory diseases such as Crohn's disease, psoriasis or multiple sclerosis. Ustekinumab blocks the IL-12 / 23 signaling pathway, thereby inhibiting the t cell-mediated immune response. Ustekinumab has been approved by FDA for the treatment of psoriasis, and was approved in China in 2017, the main indication is moderate to severe plaque psoriasis [1].

[0003] The clinical efficacy of anti-IL-12 / 23 therapy is usually related to the trough value of the therapeutic antibody, that is, the drug concentration before the next administration. There are many factors that affect the trough value, including the dose and frequency of anti-IL-12 / 23 therapy, disease activity, individual pharmacokinetics, and immune response (formation of drug-resistant antibodies, ADA) [2]. In moderate to severe cases, patients will use biological drugs such as ustekinumab for treatment. Although it has been proven that these drugs can improve the symptoms of patients, there are still some individuals who do not respond to treatment, meaning they see no improvement, or lose response over time, meaning the degree of improvement is reduced. One of the reasons for the loss of response is that the drug concentration in the patient's blood is too low. Therefore, it is an attractive research direction to measure the drug concentration to understand the status of the drug in the patient's body.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide a ustekinumab drug concentration detection kit and a preparation method, which is used for measuring the free therapeutic ustekinumab antibody concentration in human plasma and serum.

[0006] The present application provides a ustekinumab drug concentration detection kit and a preparation method, which comprises a microwell plate, a biotin-labeled anti-ustekinumab antibody, a calibrator and a quality control. The microwell plate is coated with a mouse anti-ustekinumab antibody. The biotin-labeled anti-ustekinumab antibody has biotin modified on the antibody, which is conducive to further improving the amplification of the detection signal and improving the sensitivity of the detection. The calibrator comprises seven different concentrations of ustekinumab solution. The quality control is two ustekinumab solutions with a specified concentration.

[0007] In the above scheme, preferably, the ustekinumab drug concentration detection kit further comprises a detection buffer, a substrate solution, a washing solution and a termination solution.

[0008] In the above scheme, preferably, the ustekinumab drug concentration detection kit further comprises a streptavidin-labeled horseradish peroxidase conjugate, and the working concentration of the enzyme conjugate is 0.5-2 μg / mL, and preferably 1 μg / mL.

[0009] In the above scheme, preferably, the enzyme conjugate solution is prepared by diluting the streptavidin-labeled HRP to 0.5-2 μg / mL with 0.05 M PBS (pH = 7.4) buffer.

[0010] In the above scheme, preferably, the preparation of the microwell plate coated with the mouse anti-ustekinumab antibody comprises the following steps:

[0011] (1) dilute the mouse anti-ustekinumab antibody with 10 mmol / L TRIS buffer (containing 25 mol / L EDTA);

[0012] (2) add 100 μL of the antibody solution to each well of the microwell plate, and dry in a drying chamber after overnight at 4℃.

[0013] It should be understood that the above is a preferred embodiment of the present application, and it should be pointed out that any modification, equivalent replacement, improvement, etc. made by those of ordinary skill in the art without departing from the principles of the present application should be included within the scope of protection of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The detection principle diagram of the ustekinumab drug concentration detection kit of the present application;

[0015] Figure 2 The standard curve of the detection method in Example 1 in the present application; DETAILED DESCRIPTION

[0016] The present application provides a ustekinumab drug concentration detection kit and a preparation method thereof, and the detection principle diagram is shown in Figure 1 The enzyme-linked immunoassay is used, the microwell plate is coated with anti-ustekinumab antibody, biotin-labeled anti-ustekinumab antibody and streptavidin-labeled horseradish peroxidase, which is conducive to further improving the amplification of the detection signal and improving the sensitivity of the detection.

[0017] The present application will be described in detail below in conjunction with the specific embodiments.

[0018] Example 1

[0019] (I) The present embodiment provides a ustekinumab drug concentration detection kit, and the main components thereof are as follows:

[0020] (1) Microplate

[0021] Microplate (12 rows x 8 wells) made of PS (polystyrene) pre-coated with mouse anti-ustekinumab antibody.

[0022] (2) Sample dilution buffer

[0023] 50 mmol / L TRIS buffer, 2 bottles, 100 mL per bottle, can be used directly;

[0024] (3) Calibrator

[0025] The calibrator is an ustekinumab solution prepared with artificial serum, with concentrations of 0, 5, 10, 50, 100, 200, and 400 ng / mL, each concentration is packaged in one bottle, a total of 7 bottles, labeled 1-7.

[0026] (4) Quality control

[0027] The quality control is an ustekinumab solution diluted with artificial serum, with two specified concentrations, 25 ng / mL and 100 ng / mL, labeled 1 and 2, each concentration is packaged in one bottle, a total of 2 bottles.

[0028] (5) Biotin-labeled mouse anti-ustekinumab antibody

[0029] Biotin-labeled mouse anti-ustekinumab antibody, 1 bottle, 12 mL per bottle, can be used directly.

[0030] (6) Enzyme conjugate

[0031] Enzyme conjugate is streptavidin-labeled horseradish peroxidase, 1 bottle, 12 mL per bottle, can be used directly.

[0032] (7) Substrate solution

[0033] Substrate solution is tetramethylbenzidine (TMB) solution, 1 bottle, 15 mL per bottle, can be used directly.

[0034] (8) Stop solution

[0035] 0.2M sulfuric acid solution, 1 bottle, 15 mL per bottle, can be used directly.

[0036] (II) The preparation method of the ustekinumab drug concentration detection kit described in this example is as follows:

[0037] 1. Microplate coating

[0038] (1) Dilute the mouse anti-ustekinumab antibody with 10 mM Tris buffer (containing 25 mM EDTA);

[0039] (2) Add 200 μL of antibody solution to each well of the microplate and incubate at 4°C overnight;

[0040] (3) After removing the supernatant, wash repeatedly with washing solution for 3 times;

[0041] (4) Add 200 μL of blocking solution (1% BSA) to each well of the microplate, block at room temperature for 2 hours, remove the blocking solution and dry at 37°C for standby.

[0042] 2. Biotin-labeled mouse anti-Ustekinumab antibody

[0043] Take 50 μL of 1 mg / mL biotin dissolved in DMF and add to 0.05 M PBS buffer (pH 7.2) containing 0.3 mg of mouse anti-Ustekinumab antibody, and react at room temperature for 1 hour in the dark, to cross-link biotin and mouse anti-Ustekinumab antibody by chemical reaction; dialyze the prepared biotin-labeled mouse anti-Ustekinumab antibody at 4°C overnight, and finally add the dialyzed biotin-labeled mouse anti-Ustekinumab antibody to 1% BSA and 0.05% Proclin 300 as stabilizer and preservative, and store at 4°C in a sealed manner.

[0044] 3. Preparation of enzyme conjugate

[0045] (1) Take 0.1 g of streptavidin freeze-dried powder and add to 10 mL of 10 mmol / L TRIS buffer for dissolution and mixing;

[0046] (2) Dilute HRP solid with deionized water to 5 mg / mL, and cross-link HRP and streptavidin using a simple sodium periodate method. Take the prepared streptavidin-labeled HRP, and add 1% BSA, 0.1% Tween-20 and 0.05% Proclin 300 as stabilizer and preservative, take 12 mL for dispensing, and store at 4°C in a sealed manner, and the working concentration of the enzyme conjugate is 1 μg / mL.

[0047] 4. Preparation of calibrators

[0048] Take Ustekinumab and dilute with artificial serum to prepare calibrators with concentrations of 0, 5, 10, 50, 100, 200 and 400 ng / mL, respectively, and dispense each concentration into one bottle, and store at 4°C in a sealed manner.

[0049] 5. Preparation of quality control

[0050] Dilute Ustekinumab with artificial serum to prepare quality control 1 and quality control 2, with a concentration of about 25 ng / mL for quality control 1 and a concentration of about 100 ng / mL for quality control 2, and store at 4°C in a sealed manner.

[0051] Example 2

[0052] This example is to detect the drug concentration of ustekinumab by quantitative detection.

[0053] In the first incubation step, free ustekinumab in the sample binds to the specific monoclonal anti-ustekinumab antibody coated on the microplate. After washing, peroxidase-labeled anti-ustekinumab antibody is added to detect ustekinumab bound to the microplate. Tetramethylbenzidine (TMB) is used as a substrate for peroxidase. Finally, a stop solution is added to stop the reaction. The color changes from blue to yellow. The intensity of the color is directly proportional to the concentration of free ustekinumab in the sample. According to the values of the calibrators, a dose-response curve of absorbance units (optical density, OD) versus concentration is generated. The concentration of free ustekinumab in the sample is directly determined on the curve.

[0054] 1. Standard curve

[0055] The calibrators and quality control samples are added to the microplate at 50 μL per well, followed by the addition of 50 μL of detection buffer. The plate is sealed with a sealing film and incubated for 1 hour at room temperature (15-30°C) on a horizontal microplate shaker. The plate is washed 3 times. Then 100 μL of biotin-labeled mouse anti-ustekinumab antibody is added to each well, the plate is sealed with a sealing film, and incubated for 1 hour at room temperature (15-30°C) on a horizontal microplate shaker. The plate is washed 3 times. Then the enzyme conjugate is added and incubated for 30 minutes, and the plate is washed 3 times. Finally, the substrate TMB is added and incubated for 15 minutes, and the color development reaction is stopped by adding the stop solution HCL. The absorbance value is read on a microplate reader at a wavelength of 450 nm. The results are shown in Table 1.

[0056] Table 1 OD values of different concentrations of ustekinumab

[0057] Concentration ng / mL OD1 OD2 0 0.042 0.037 5 0.145 0.137 10 0.213 0.207 50 0.512 0.524 100 0.823 0.831 200 1.567 1.552 400 2.863 2.857 Quality Control 1 0.294 0.287 Quality Control 2 0.812 0.825

[0058] According to the above detection results, the concentration value is taken as the abscissa and the OD value is taken as the ordinate, and the curve fitting is performed to obtain the curve equation: y = 0.0069x + 0.1215, R2 = 0.9978, and the curve is shown in Figure 2 .

[0059] 2. Specificity analysis

[0060] The specificity of the antibody is determined by detecting cross-reactivity. Cross-reactivity is detected with components that have structural similarity to ustekinumab antibodies, and the results are shown in Table 2. It is found that there is no cross-reactivity.

[0061] Table 2 Specificity test results

[0062]

[0063] 3. Accuracy

[0064] Accuracy refers to the consistency between the detection result and the true value of the measured object. Therefore, a known concentration of ustekinumab is added to two different serum samples, and the results are shown in Table 3. It can be seen that the recovery rate is within 80-120%, indicating that the recovery rate is good, and the kit accuracy is high.

[0065] Table 3 Accuracy test results

[0066]

[0067]

[0068] 4. Comparison with other methods

[0069] The kit of the present embodiment is compared with a certain brand of commercially available product, as shown in Table 4.

[0070] Table 4 Comparison of the kit of the present embodiment with a certain brand of commercially available product

[0071] The kit of this example A certain brand commercially available Linear range 5-400 ng / mL 10-300 ng / mL Sample volume 50 μL 100 μL Limit of detection 1.231 1.495 ng / ml

[0072] In summary, the detection kit provided by the present application has the advantages of small sample amount, wide linear range and high sensitivity, and has good application prospect.

[0073] The present application is not limited to the above best embodiment, and any person should know that the structural changes made under the inspiration of the present application fall within the protection scope of the present application.

[0074] References:

[0075] [1] Zhao X, Wang YD. Clinical research progress of ustekinumab in the treatment of Crohn's disease [J]. Chinese General Medicine, 2022, 25(27): 3352-3357

[0076] [2] Chiu HY, Chu TW, Cheng YP, Tsai TF. The association between clinical response to ustekinumab and immunogenicity to ustekinumab and prior adalimumab [J]. PLoS One, 2015, 10: 1-10.

Claims

1. A reagent kit for detecting ustekinumab drug concentration, characterized in that, The assay comprises a microplate, biotin-labeled anti-ustekinumab antibody, calibrators, and quality controls. The microplate is coated with mouse anti-ustekinumab antibody. The biotin-labeled anti-ustekinumab antibody is an antibody modified with biotin, which helps to further amplify the detection signal and improve the detection sensitivity. The calibrators include seven different concentrations of ustekinumab solution. The quality controls consist of two calibrated concentrations of ustekinumab solution.

2. The ustekinumab drug concentration detection kit according to claim 1, characterized in that, The ustekinumab drug concentration detection kit also includes a detection buffer, substrate, washing solution, and stop solution.

3. The ustekinumab drug concentration detection kit according to claim 1, characterized in that, The pre-detection kit also contains an enzyme conjugate, which is a streptavidin-labeled horseradish peroxidase conjugate, and the working concentration of the enzyme conjugate is 0.5-2 μg / mL, preferably 1 μg / mL.

4. The ustekinumab drug concentration detection kit according to claim 1, characterized in that, The enzyme conjugate solution was prepared by using streptavidin-labeled HRP, which was diluted to 0.5-2 μg / mL with 0.05M PBS (pH=7.4) buffer.

5. The ustekinumab drug concentration detection kit according to claim 1, characterized in that, The preparation of the microplate coating with mouse anti-ustekinumab antibody includes the following steps: (1) Dilute the mouse anti-ustekinumab antibody with 10 mM Tris buffer (containing 25 mM EDTA); (2) Add 200 μL of antibody solution to each well of the microplate and incubate overnight at 4°C; (3) After removing the supernatant, rinse repeatedly with cleaning solution 3 times; (4) Add 200 μL of blocking solution (1% BSA) to each well of the microplate, block at room temperature for 2 hours, remove the blocking solution, and dry at 37 degrees for later use.

6. The ustekinumab drug concentration detection kit according to claim 1, characterized in that, The preparation of the biotin-labeled mouse anti-ustekinumab antibody includes the following steps: (1) Take 50 μL of biotin dissolved in DMF at 1 mg / ml and add it to 0.05 M PBS buffer (pH 7.2), wherein the PBS buffer contains 0.3 mg of mouse anti-ustekinumab antibody. React at room temperature in the dark for 1 hour to crosslink biotin with mouse anti-ustekinumab antibody through chemical reaction. (2) The prepared biotin-labeled mouse anti-ustekinumab antibody was dialyzed overnight at 4°C; (3) The biotin-labeled mouse anti-ustekinumab antibody after dialysis was added to 1% BSA and 0.05% Proclin300 as stabilizers and preservatives, and stored in a sealed container at 4°C.

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